Hierarchical silica lamella for magnetic nucleic acid extraction

ABSTRACT

Disclosed herein is a novel method to fabricate magnetic silica nanomembranes using thin polymer cores based on silica deposition and self-wrinkling induced by thermal shrinkage. These micro- and nano-scale structures have vastly enlarged the specific area of silica, thus the magnetic silica nanomembranes can be used for solid phase extraction of nucleic acids. The magnetic silica nanomembranes are suitable for nucleic acid purification and isolation and demonstrated better performance than commercial particles in terms of nucleic acid recovery yield and integrity. In addition, the magnetic silica nanomembranes may have high nucleic acid capacity due to significantly enlarged specific surface area of silica. Methods of use and devices comprising the magnetic silica nanomembranes are also provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application ofPCT/US2016/014920 filed 26 Jan. 2016, which claims priority to U.S.Provisional Application No. 62/108,288 filed 27 Jan. 2015, the entiredisclosures of which are hereby incorporated by reference in theirentireties.

BACKGROUND

Isolation of nucleic acids from samples such as cells, tissues, plants,bacteria, viral particles, blood, serum, or plasma may be an importantstep for genetic analysis. Conventionally, liquid phase extractiontechniques, such as phenol/chloroform precipitation, are widely used.Although these approaches may yield nucleic acids of high quality, theycan be laborious, time-consuming, and highly operator-dependent. Solidphase extraction techniques are a popular alternative. They are oftenthe methods of choice when processing large numbers of samples. Commonlyused solid-phase substrates include, for example, silica spin columnsand silica magnetic particles that may provide large surface areas fornucleic acid binding. These porous matrices and micro/nano particles,however, may induce nucleic acid shearing as a result of flow andparticle mixing, leading to decreased nucleic acid integrity.

The dominant methods of nucleic acid extraction, such as DNA extraction,have remained remarkably unchanged since spin columns and magneticmicroparticles were first introduced. While methods such as spin columnsand magnetic microparticles are fast and easy, the shear forces imposedby these methods may fragment the nucleic acids and may be incapable ofsufficient nucleic acid quality for the new generation of long readlength sequencing and genome mapping technologies. As such, there existsan unmet need to develop novel separation materials and methods thatallow for easier isolation and purification of nucleic acids frombiological samples.

Thermoplastic substrates have previously been disclosed that contain ahierarchical structure of microscale folds layered with nanoscale silicalamella that are easily fabricated using an inexpensive heat-shrinkablepolyolefin (PO) film. This nanomembrane can be fine-tuned to create anon-porous, high surface area binding substrate capable of capturingvast amounts of nucleic acids without imparting nucleic acid fragmentingshear forces. By minimizing fragmentation, it may be possible to biasnucleic acid binding away from a prone conformation towards a tentacleconformation, increasing binding capacity to, for example, about 100 toabout 1,000,000 times greater than previously reported for silicamicroparticles. Furthermore, the silica nanomembranes use a simple bind,wash, and elute protocol that combines the ease of column and beadextraction with the performance of phenol-chloroform, resulting innucleic acid yields that can be about 10 times greater than eithercolumns or magnetic beads and nucleic acids of high purity and highmolecular weight.

To facilitate extraction of large amounts of high quality, highmolecular weight nucleic acids from cells, tissues, and body fluids,disclosed herein is a magnetic silica nanomembrane material made bydepositing a magnetic component on or embedded in a thermoplasticsubstrate in addition to at least one silica layer. The magnetic silicananomembrane enables extraction to proceed analogously to a magneticprocess whereby a magnet can be used to draw the nanomembrane to a sideor bottom of a container, such as a test tube, thereby facilitatingpipetting and washing without disturbing the nanomembrane or the boundnucleic acids.

Disclosed herein are inexpensive magnetic thermoplastic nanomembranematerials that use a hierarchical layering of micro- and nanoscalesilica lamella and a magnetized layer to create a high surface area andlow shear substrate capable of capturing vast amounts of high molecularweight nucleic acid without fragmentation.

BRIEF SUMMARY

Disclosed herein are magnetic silica nanomembranes comprising a polymercore having a first surface and a second surface; at least one silicondioxide layer disposed over the polymer core, the silicon dioxide layercomprising at least one surface morphology chosen from a plurality of(a) microscale silica structures and (b) nanoscale silica structures;and at least one magnetic component.

In certain embodiments, the at least one magnetic component comprises atleast one magnetic material chosen from diamagnetic materials,paramagnetic materials, ferrimagnetic materials, and ferromagneticmaterials, and in certain embodiments, the at least one magneticcomponent comprises at least one magnetic material chosen from iron,nickel, cobalt, magnetite, hematite, maghemite, and alloys of magneticmaterials such as steel, alperm, permalloy, fernico, sendust, cunife,and alnico.

In certain embodiments, the at least one magnetic component is chosenfrom at least one layer of magnetic material disposed over the polymercore, at least one magnetic material embedded within the polymer core,and at least one polymer core being magnetic. In certain exemplaryembodiments, the at least one magnetic component is a magnetic layerhaving a thickness ranging from about 5 nm to about 10 μm thick.

In certain embodiments, the at least one magnetic component comprises alayer of magnetic material disposed over the at least one silicondioxide layer. In certain embodiments disclosed herein, the at least onesilicon dioxide layer is disposed over the first surface of the polymercore and the at least one magnetic component is a magnetic layerdisposed over the second surface. In certain embodiments, a firstsilicon dioxide layer is disposed over the first surface of the polymercore, the at least one magnetic component is a magnetic layer disposedover the second surface, and a second silicon dioxide layer is disposedover the at least one magnetic component. In various other exemplaryembodiments, a first silicon dioxide layer is disposed over the firstsurface of the polymer core, the at least one magnetic component is amagnetic layer disposed over the second surface, a second silicondioxide layer is disposed over the at least one magnetic component, anda third silicon dioxide layer is disposed between the first surface ofthe polymer core and the at least one magnetic component. In furtherembodiments, a first silicon dioxide layer is disposed over a firstsurface of the polymer core, at least one magnetic component is disposedover a second surface of the polymer core, a second silicon dioxidelayer is disposed over the at least one magnetic component, a thirdsilicon dioxide layer is disposed between the second surface of thepolymer core and the at least one magnetic component, a second magneticcomponent is disposed over the first silicon dioxide layer disposed overthe first surface of the polymer core, and a fourth silicon dioxidelayer is disposed over the second magnetic component. In variousembodiments, a second magnetic component is disposed between the firstsurface of the polymer core and the at least one silicon dioxide layer.In certain embodiments, the magnetic nanomembrane disclosed hereinfurther comprises a passivation layer disposed over the at least onemagnetic component, and in certain embodiments, the at least one silicondioxide layer has a thickness ranging from about 2 nm to about 500 nmthick.

In certain embodiments disclosed herein, the at least one silicondioxide layer is deposited using a deposition method chosen fromelectron beam evaporation, sputtering, chemical vapor deposition, plasmaenhanced chemical vapor deposition, electroplating, atomic layerdeposition, chemical solution deposition, and spin coating. In variousother embodiments disclosed herein, the magnetic silica nanomembranesdisclosed herein further comprise a surface functionalization chosenfrom at least one of aminopropyl groups, chloropropyl groups, octadecylgroups, octyl groups, quaternary ammonium groups, diethlylaminoethylgroup, sulfonic acid groups, phenyl groups, chitosan, biotin,streptavidin, antibodies, proteins, lipids, polyethylene glycol, andenzymes. In certain exemplary embodiments, the polymer core of themagnetic silica nanomembranes disclosed herein comprises at least onethermoplastic material chosen from polymethyl methacrylate,polycarbonate, polystyrene, cyclic polyolefin polymers, polypropylene,polyvinyl chloride, polyethylene, fluorinated ethylene propylene,polytetrafluoroethylene, and polyvinylidene fluoride.

Also disclosed herein are methods for extracting nucleic acids from asample, the methods comprising obtaining a sample comprising nucleicacids; contacting the sample with at least one magnetic nanomembrane,the at least one magnetic nanomembrane comprising at least one silicondioxide layer and at least one magnetic component; allowing the nucleicacids in the sample to adsorb onto the at least one magneticnanomembrane; manipulating the at least one magnetic nanomembrane usinga magnet; and desorbing the nucleic acids from the at least one magneticsilica nanomembrane to obtain extracted nucleic acids from the sample.

In certain embodiments of the methods disclosed herein for extractingnucleic acids from a sample, the nucleic acids are chosen from DNA, RNA,and mixtures of DNA and RNA. In certain embodiments, the nucleic acidsare dispersed in a supernatant comprising a lysis buffer, or, in certainembodiments, the nucleic acids are dispersed in a reaction solution.

In certain embodiments of the methods disclosed herein, manipulating theat least one magnetic nanomembrane comprises holding the magneticnanomembrane in a desired position with the magnet while removing thesolution from contact with magnetic nanomembrane, and magneticallyreleasing, and in certain embodiments, manipulating the at least onemagnetic nanomembrane comprises transferring the magnetic nanomembranewith the magnet and magnetically releasing the at least one magneticnanomembrane after transfer.

In certain embodiments of the methods disclosed herein, washing the atleast one magnetic nanomembrane comprises a) contacting the at least onemagnetic nanomembrane with a wash solution; and b) manipulating the atleast one magnetic nanomembrane to separate the wash solution from themagnetic nanomembrane. In various embodiments, the methods disclosedherein may further comprise repeating the process of steps a) and b) oneor more times.

In certain embodiments of the methods disclosed herein, desorbing thenucleic acids comprises contacting the sample with an elution solutionand releasing the at least one magnetic nanomembrane into the elutionsolution, and in certain embodiments, the extracted nucleic acids havean average length of at least about 100 kilobases. Also disclosed hereinare embodiments wherein the method for extracting nucleic acids from asample is performed in an automated manner by a robotic instrument.

Further areas of applicability of the embodiments disclosed herein willbecome apparent from the detailed description provided hereinafter. Itshould be understood that the detailed description and specific examplesare intended for purposes of illustration only and are not intended tolimit the scope of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will become more fully understood from thedetailed description and the accompanying drawings, wherein:

FIG. 1 is a diagram illustrating a fabrication process for a magneticsilica nanomembrane comprising a polymer core, a layer of silicondioxide, and a layer of iron.

FIG. 2 illustrates a fabrication process for a magnetic silicananomembrane comprising a polymer core, multiple layers of silicondioxide, and a layer of iron.

FIG. 3 is a flow chart illustrating an exemplary method for DNAextraction using a magnetic silica nanomembrane.

FIG. 4 is a photograph of a magnetic nanomembrane disc being attractedto the side of a microcentrifuge tube using a magnet.

FIG. 5 is a bar graph comparing the DNA yield for extraction using amagnetic silica nanomembrane versus phenol-chloroform, Qiagen Minispincolumn, and Qiagen magnetic particle technology.

FIG. 6 is a graph illustrating the UV absorbance versus the wavelengthfor DNA extracted from MCF-7 cells using phenol chloroform, magneticsilica nanomembrane, magnetic bead microparticles, and a spin column.

FIG. 7 is a pulsed field gel image illustrating the size of genomic DNAextracted from MCF-7 cells using phenol chloroform, magnetic silicananomembrane, magnetic bead microparticles, and a spin column.

FIG. 8 is a bar graph illustrating the yield for total nucleic acidextracted from whole human blood using a magnetic nanomembrane and anon-magnetic nanomembrane.

FIG. 9 is a photograph showing magnetic nanomembranes captured by themagnetic rods on the robotic arm of a Thermo Scientific KingFisher™ DuoPrime automated DNA extraction system.

FIG. 10 is a bar graph showing the DNA recovery from eight samples runon an automated DNA extraction system.

FIG. 11 is a schematic illustration of automated DNA extraction using amagnetic nanomembrane and a magnetic rod actuated instrument.

FIG. 12 is a schematic illustration of an exemplary transfer of magneticnanomembranes between solutions in an automated DNA extraction using amagnetic rod actuated instrument.

FIG. 13 is a scanning electron microscope (SEM) image illustrating thesurface topology of a magnetic silica nanomembrane. The inset shows aregion at higher magnification.

FIGS. 14A, 14B, and 14C illustrate various exemplary embodiments ofmagnetic silica nanomembranes comprising a polymer core, at least onelayer of silicon dioxide, and at least one magnetic component.

DETAILED DESCRIPTION

The following description of various embodiments is merely exemplary innature and is in no way intended to limit the disclosure, itsapplications, or uses thereof.

As used throughout, ranges are used as shorthand for describing each andevery value that is within the range. Any value within the range can beselected as the terminus of the range. In addition, all references citedherein are hereby incorporated by reference in their entireties. In theevent of a conflict in a definition in the present disclosure and thatof a cited reference, the present disclosure controls.

As used herein, the term “one or more of” with respect to a listing ofitems such as, for example, A and B, means A alone, B alone, or A and B.The term “at least one of” is used to mean one or more of the listeditems can be selected.

Unless otherwise specified, all percentages and amounts expressed hereinand elsewhere in the specification should be understood to refer topercentages by weight. The amounts given are based on the active weightof the material.

Disclosed herein are nucleic acid extraction methods based on novel andrelatively inexpensive hierarchical magnetic silica nanomembranes. Themagnetic silica nanomembranes disclosed herein are polymer substratescontaining a hierarchical topography of microscale and/or nanoscalesilica structures and at least one magnetic component. As used herein, amicroscale structure is understood as having characteristic dimensionsof less than or about 1000 μm, such as less than or about 500 μm, lessthan or about 200 μm, or less than or about 100 μm. As used herein, ananoscale structure is understood as having characteristic dimensions ofless than or about 1000 nm, such as less than or about 500 nm, less thanor about 200 nm, or less than or about 100 nm. Unlike silica magneticbeads and columns, which impart DNA/RNA fragmenting shear forces, thenon-porous silica nanomembrane substrates disclosed herein can bind andrelease DNA/RNA without fragmenting it, achieving increased DNA/RNA size(up to the Mb range), which may exceed phenol-chloroform extractions,considered by many to be a gold standard in the art. Moreover, themethods disclosed herein for nucleic acid extraction using magneticsilica nanomembranes may in many respects be simpler than magnetic beadsand columns. Furthermore, the methods disclosed herein may have anextraction yield that is at least about five to about thirty foldgreater than known methods employing magnetic beads and/or columns.

The magnetic silica nanomembranes disclosed herein may be used toextract nucleic acids from cultured cells, tissues, bacteria, virus,plant cells, whole blood, serum, buffy coat, plasma, urine, sputum,stool, pleural effusion, cerebral spinal fluid, ductal lavage,formalin-fixed paraffin embedded (FFPE) tissue samples, or other nucleicacid containing materials. It is understood to one skilled in the artthat modifications to the extraction buffer systems may be necessary toaccommodate different sample types.

As used herein, the term “silica nanomembranes” means three-dimensionalconformations of the silica on a polymer core, which can comprisestructures such as micro-wrinkles, nano-wrinkles and silica flakes,ranging from tens of nanometers to micrometers in size. The termslamella, wrinkle, fold, flake, chip, and the like are descriptive termsused to describe the appearance of silica structures on the nanomembranesurface. As one of ordinary skill in the art would appreciate, thetopography adopted by the silica at the microscale and/or nanoscalelevel may vary according to various factors, including, for example, theamount of silicon dioxide deposited and the unique conformation adoptedby the polymer core during shrinkage, and the embodiments disclosedherein are not limited by the topography adopted by the at least onesilicon dioxide layer that is deposited over the polymer core.

The term “silica” as used herein means silicon oxide, silicon dioxideand silicon dioxide derivatives, such as SiO₂ crystals and other formsof SiO₂, for example diatoms composed of SiO₂, zeolites, amorphoussilicon dioxide, glass powder, silicic acid, waterglass, borosilicate,and also aluminum silicates and activated silicates.

The term “sample” or “biological sample” as used herein refers to anysample that comprises cells or cellular material, such as cells, frozencell pellets, fixed cells, feces/stool, buffy coat (i.e., white bloodcell fraction of blood), ascites, swabs, such as cheek or throat swabs,cervical swabs, sputum, organ punctates, sperm, tissue samples, fixedtissue samples, tissue sections of fixed or nonfixed tissue samples,such as frozen sections and paraffin sections, such as formalin-fixedparaffin sections, tumor material, biopsy samples, blood samples, suchas whole blood or blood fractions, cell suspensions, and in the broadestsense all samples that comprise cellular constituents, wherein bothintact cells and cell constituents shall be comprised. Furthermore, theterm also comprises other nucleic acid-containing, biological materials,such as, for example, blood serum or blood plasma, such asvirus-containing serum or plasma, HIV- and HCV-infected serum samples,secretions, CSF, bile, lymph fluid, and urine. Similarly, it can benucleic acid-containing materials that originate from biochemical orbiotechnological processes and are to be subsequently purified.

As used herein, the term “nucleic acid” includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoramidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. In certain embodiments the nucleic aciddoes not comprise any insertions, deletions, inversions, and/orsubstitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

In certain embodiments, the nucleic acids disclosed herein arerecombinant. As used herein, the term “recombinant” refers to (i)molecules that are constructed outside living cells by joining naturalor synthetic nucleic acid segments to nucleic acid molecules that canreplicate in a living cell, or (ii) molecules that result from thereplication of those described in (i) above. For purposes herein, thereplication can be in vitro replication or in vivo replication.

As used herein, the term “polymer” means any polymer substrate that iscapable of heat shrinkage. In some embodiments, the polymers arethermoplastic polymers. As used herein, the term “thermoplastic” means apolymer that becomes pliable or moldable above a specific temperatureand returns to a solid state upon cooling. Thermoplastics can include,for example, polymers such as polymethyl methacrylate (PMMA),polycarbonate, polystyrene (PS), and cyclic polyolefin (PO) polymers.

The silica nanomembranes used herein may be manufactured using commonlyused polymer substrates, including, for example, pre-stretchedthermoplastics, such as PMMA, polycarbonate, PS, and cyclic PO polymers.Other exemplary polymer substrates that may be used as the polymer coreof the silica nanomembrane include, for example, polyvinyl chloride,polyethylene, polypropylene, fluorinated ethylene propylene,polytetrafluoroethylene, and polyvinylidene fluoride. In certainembodiments, silica may be deposited over shrinkable PO films. Afterincubation at elevated temperatures, the polymer film shrinks, and thesilica forms nanostructures due to the aforementioned mechanism.

In certain embodiments, the polymer core of the magnetic silicananomembrane has a shrunken thickness ranging from about 5 μm to about 5mm. In certain embodiments, the polymer core of the magnetic silicananomembrane has a pre-shrunken thickness ranging from about 5 μm toabout 500 mm.

In certain embodiments disclosed herein, the magnetic silicananomembrane may be fabricated by coating the polymer core, such as apolyolefin polymer film, with at least one layer of silicon dioxide. Incertain embodiments, the at least one layer of silicone dioxide mayrange in thickness from about 2 nm to about 500 nm, such as about 50 nmto about 200 nm, about 75 nm to about 150 nm, or about 100 nm. In oneembodiment, a 20 μm thick polyolefin film is coated on one side with a100 nm thick layer of silicon dioxide using e-beam evaporation. Thesilicon dioxide may be deposited directly on the polymer core, or,alternatively, in certain embodiments the silicon dioxide may bedeposited over the polymer core when the polymer core is first coatedwith at least one magnetic component, at least one inert layer, and/orat least one additional layer of silicon dioxide.

After the polymer core has been coated with at least one layer ofsilica, the other side of the polymer core may then be coated with atleast one magnetic component, or, in certain embodiments, a second layerof silica. In certain embodiments, the other side of the silica-coatedpolymer core is then coated with at least one magnetic component, and incertain embodiments the other side of the silica-coated polymer core isfirst coated with a second silica layer, wherein the second silica layeris then coated with at least one magnetic component.

In certain embodiments disclosed herein, the other side of the polymercore, such as the polyolefin film, may then coated with a magneticcomponent. In certain embodiments, the magnetic component may be amagnetic layer ranging in thickness from about 15 nm to about 200 nm,such as about 30 nm to about 100 nm, about 100 nm or about 30 nm thick.In certain embodiments, the magnetic component may be embedded withinthe polymer core. In certain embodiments, the polymer core may becomprised of a material that is intrinsically magnetic.

In certain embodiments, a polymer core, such as a polyolefin filmranging in thickness from about 10 μm to about 100 μm, may be coated onone side with a layer of silicon dioxide. The other side of the polymercore may then be coated with a magnetic layer, such as a 20 nm to 100 nmthick magnetic layer. The coated polymer core substrate may then be heatshrunk, for example in an oven. This creates a nanomembrane with micro-and nanoscale silica structures on one side and a magnetic layer on theother side that can then be used for magnetic nucleic acid extraction.In certain embodiments, the magnetic layer may also have nanoscale andmicroscale structures created by the created during the fabricationprocess. FIG. 1 is a flow chart illustrating an exemplary fabricationprocess according to embodiments disclosed herein. As shown in FIG. 1,at least one silicon dioxide layer 101 may be deposited over a firstsurface of a polymer core 100, such as a polyolefin film. Next, at leastone magnetic component 102, such as iron, may be deposited over a secondsurface of the polymer core 100 before the nanomembrane is heat shrunk.

In certain embodiments, the nanomembrane coated with silicon dioxide onone side may be alternately coated with iron and silicon dioxide on theother side, and then the coated polyolefin substrate may be heat shrunk.This creates a nanomembrane with micro- and nanoscale silica structureson both sides. The back side silica structures comprise a ferromagneticiron layer covered by silica such that magnetic manipulation can beperformed and nucleic acids can bind to both sides of the nanomembrane.The silica covering the iron acts as a binding surface for nucleic acidsand protects the iron from reacting with buffer components. FIG. 2 is aflow chart illustrating various exemplary fabrication processesaccording to embodiments disclosed herein. As shown in FIG. 2, at leastone silicon dioxide layer 201 may be deposited over a first surface of apolymer core 200, such as a polyolefin film. Next, at least one magneticcomponent 202, such as iron, may be deposited over a second surface ofthe polymer core 200. In certain embodiments, a second silicon dioxidelayer 201 may be deposited over the at least one magnetic component 202before the nanomembrane is heat shrunk.

Alternatively, as shown in FIG. 2, at least one silicon dioxide layer201 may be deposited over a first surface of the polymer core 200, and asecond silicone dioxide layer 201 may be deposited over a second surfaceof the polymer core 200. Next, at least one magnetic component 202, suchas iron, may be deposited over the second silicon dioxide layer 201.Finally, a third silicon dioxide layer 201 may be deposited over the atleast one magnetic component 202 before the nanomembrane is heat shrunk.

In certain embodiments of the disclosure, a first silicon dioxide layermay be deposited on a first surface of the polymer core, and at leastone magnetic component may be deposited on the second surface of thepolymer core, wherein a second silicon dioxide layer may be depositedover the at least one magnetic component. Furthermore, in certainembodiments, a second magnetic component may be deposited between thefirst silicon dioxide layer and the first surface of the polymer core.

As shown in FIG. 14A, in certain embodiments, at least one layer ofsilicon dioxide 1401 may be deposited over a first surface of thepolymer core 1400 and at least one magnetic component 1402 may bedeposited over the at least one silicon dioxide layer 1401. In certainembodiments, a second silicon dioxide layer 1401 may be deposited overthe at least one magnetic component 1402. Optionally, no layers may bedeposited on a second surface of the polymer core 1400, such that, forexample, the nanomembrane contains a polymer core 1400, a first layer ofsilicon dioxide 1401, a magnetic component 1402, and a second layer ofsilicon dioxide 1401.

Alternatively, in certain embodiments and as shown in FIG. 14A, at leastone layer of silicon dioxide 1401 may be deposited over a first surfaceof the polymer core 1400 and at least one second layer of silicondioxide 1401 may be deposited over a second surface of the polymer core1400. Next, at least one magnetic component 1402 may be deposited overthe at least one silicon dioxide layer 1401 and a second magneticcomponent 1402 may deposited over the second silicon dioxide layer 1401.Finally, in various optional embodiments illustrated in FIG. 14A, athird layer of silicon dioxide 1401 may be deposited over the at leastone magnetic component 1402, and, optionally, a fourth layer of silicondioxide 1401 may be deposited over the second magnetic component 1402.

As shown in FIG. 14B, in certain embodiments, at least one magneticcomponent 1402 may be deposited over a first surface of a polymer core1400, and at least one layer of silicon dioxide 1401 may be depositedover the at least one magnetic component. In various optionalembodiments, no layers may be deposited on a second surface of thepolymer core 1400, such that, for example, the nanomembrane contains apolymer core 1400, a magnetic component 1402, and a silicon dioxidelayer 1401.

In certain other embodiments illustrated in FIG. 14B, at least one firstmagnetic component 1402 may be deposited over a first surface of apolymer core 1400 and at least one layer of silicon dioxide 1401 may bedeposited over a second surface of the polymer core 1400. Next, a secondmagnetic component 1402 may be deposited over the at least one layer ofsilicon dioxide 1401. Optionally, in certain embodiments, a second layerof silicon dioxide 1401 may be deposited over the second magneticcomponent 1402. In certain other embodiments illustrated in FIG. 14B, athird layer of silicon dioxide 1401 may be deposited over the at leastone first magnetic component 1402.

As shown in FIG. 14C, in certain embodiments, at least one firstmagnetic component 1402 may be deposited on a first surface of a polymercore 1400 and at least one second magnetic component 1402 may bedeposited on a second surface of the polymer core 1400. The magneticcomponents may be the same or different. In certain embodiments, atleast one layer of silicon dioxide 1401 may be deposited over the atleast one first magnetic component 1402. In certain exemplaryembodiments, optionally a second layer of silicon dioxide 1401 may bedeposited over the second magnetic component 1402.

In various other embodiments, a polyolefin film may be coated on oneside with a silicon dioxide and then heat shrunk. Next, a magnetic layermay be deposited on the other side of the nanomembrane after heatshrinking. In alternate embodiments disclosed herein, after thepolyolefin film is heat shrunk, the at least one magnetic component maybe coated with silicon dioxide to act as a passivation layer.

In certain embodiments disclosed herein, the magnetic component can bediamagnetic, paramagnetic, ferromagnetic, or ferrimagnetic. In variousembodiments, the magnetic component is paramagnetic such that thenanomembranes do not stick to one another. In various embodiments, themagnetic component can be made from iron, nickel, cobalt, magnetite,hematite, maghemite, magnetic alloys such as steel, permalloy, andalnico, or any such material.

In certain exemplary embodiments, the magnetic material may have a highmagnetic susceptibility such that a thinner layer of the magneticmaterial can be deposited while achieving sufficient magnetic force topull the nanomembrane while in solution. A thicker layer may result in alarger magnetic pulling force for a given applied magnetic field. Forefficient magnetic manipulation, sufficient magnetic force is necessaryto overcome viscous forces, surface tension, buoyancy, and the like. Inone embodiment, the magnetic layer can vary in thickness from about 2 nmto about 10 μm.

In certain embodiments the magnetic material may be chemically andbiologically inert such that it does not degrade, leech, or adverselyreact with buffer components or biological materials. The magneticmaterial may also not be highly stressed and not disrupt the formationof the silica lamella structures that are necessary for nucleic acidextraction.

After deposition, the coated polymer core is then heat shrunk in anoven. This creates a nanomembrane with micro- and nanoscale silicastructures, such as lamella, on one side and a magnetic layer on theother side, which can then be used for magnetic nucleic acid extraction.

As the polymer film shrinks, differences in film stress create ahierarchical structure of microscale folds layered with nanoscale silicalamella that can be fine tuned via silicon dioxide deposition thickness.

In various embodiments disclosed herein, the polymer core may be coatedon one side with a first layer of silicon dioxide. The other side of thepolyolefin film may then alternately coated with a second layer ofsilicon dioxide, followed by a layer of a magnetic material on top ofthe second layer of silicon dioxide, and a third layer of silicondioxide on top of the layer of the magnetic material. The coated polymersubstrate may then be heat shrunk, for example in an oven. In certainembodiments the coated polymer substrate may be heat shrunk. Thiscreates a nanomembrane with micro- and nanoscale silica lamella on bothsides. The backside silica lamella comprises a magnetic centersandwiched by silica such that magnetic manipulation can be performedand nucleic acids can bind or adsorb to both sides of the nanomembrane.The silica enveloping both sides of the magnetic material acts as abinding surface for nucleic acids and protects the magnetic materialfrom reacting with buffer components. In certain embodiments, themagnetic material may be iron. In other embodiments, the magneticmaterial may be an alloy such as steel, alnico, permalloy, alperm,fernico, sendust, cunife, or the like.

In another embodiment, the polymer core may be coated on one side with afirst silica layer. The other side of the polymer film may then bealternately coated with a layer of a magnetic material and a secondsilica layer. The coated polymer substrate may then be heat shrunk.

In another embodiment, a polymer core may be coated on one side with asilica layer and then heat shrunk. Next, a magnetic layer may bedeposited on the other side of the nanomembrane after heat shrinking.

In the embodiments disclosed herein, the deposition of the layers may bedone by any means known in the art. In certain embodiments, the layerscan be deposited by thermal evaporation, electron beam evaporation,sputtering, chemical vapor deposition, plasma enhanced chemical vapordeposition, electroplating, atomic layer deposition, chemical solutiondeposition, spin coating, or any other deposition method. In oneembodiment, the layers can be deposited by electron beam evaporation.

In other embodiments, the magnetic layer may be passivated by depositinganother layer or coating to make it more chemically or biologicallyinert.

In certain embodiments disclosed herein, the silica comprising thesilica nanomembranes may be derivatized or functionalized with othercompounds or components known in the art to provide desirable chemical,physical, or electronic properties or to perform specific functions suchas to promote or prevent adsorption or binding and facilitatebiochemical reactions. In some embodiments, the silica can bederivatized with aminopropyl groups, chloropropyl groups, octadecylgroups, octyl groups, quaternary ammonium groups, diethlylaminoethylgroup, sulfonic acid groups, phenyl groups, biotin, streptavidin,antibodies, proteins, lipids, chitosan, or enzymes.

In various embodiments, the magnetic component may be covered by asilica layer to enable nucleic acid binding on both sides of thenanomembrane.

In some embodiments, the nanomembrane is heated at a temperature rangingfrom about 100° F. to 500° F., such as about 200° F. to about 400° F.,or about 250° F. to about 300° F. In some embodiments the nanomembraneis heated for a time ranging from about 10 seconds to about 10 minutes,such as about 1 minute to about 5 minutes, or about 2 minutes to about 3minutes. In some embodiments, stress may be applied to the nanomembraneto control the shrink rate and direction.

In one embodiment, the nanomembrane may be shaped to specific dimensionsafter heat shrinking by cutting or punching. In certain embodiments, forexample, a disk of the nanomembrane may formed using a punch after heatshrinking, such as a disk ranging is diameter from about 1 μm to severalmeters, such as about 1 mm to about 6 mm. In another embodiment, thenanomembrane may be shaped to specific dimensions before heat shrinkingby cutting or punching.

The hierarchical pattern on the silica nanomembranes disclosed herein isbased on the thermally induced surface wrinkling of heat-shrinkablepolymer film deposited with silica. The use of surface wrinkles causedby shrinking or swelling with a pre-stretched soft polymer substratecoated thin film of metals is a simple and low-cost method to fabricatenanomaterials. Due to different shrinkage or expansion coefficientsbetween the polymer substrate and the stiff film, stress will accumulatewithin the film and eventually lead to spontaneous surface wrinkling.

The exact nanostructures formed in the process of making the magneticsilica nanomembranes depends on the thickness of the coating or layer ofsilicon dioxide and the magnetic material being deposited. As the silicalayer gets thicker, the specific surface area of the silica nanomembranemay be greatly enhanced, and, concomitantly, the nucleic acid bindingcapacity increases. Thus, the magnetic silica nanomembranes disclosedherein may have higher nucleic acid recovery yield compared tocommercial silica columns and magnetic particles. The magnetic silicananomembranes disclosed herein may be able to extract nucleic acid, suchas DNA, from cultured human cells with high yield and high qualitycompared to other known methods, such as the phenol-chloroform method.

The magnetic silica nanomembranes disclosed herein can be fabricatedinto any suitable shape for specific purposes, such as planar or in abead conformation. In certain embodiments, the magnetic silicananomembranes can be circular, square, or any particular shape,including irregular, novelty, and three-dimensional shapes. In oneembodiment, the magnetic silica nanomembranes disclosed herein can becircular and can fit into a test tube. In certain embodiments, themagnetic silica nanomembranes can be adapted to fit into a column orpipette tip for flow-through analysis or any other apparatus capable ofholding a sample. In certain embodiments, the magnetic silicananomembranes can be folded, bent, or attached together into athree-dimensional shape.

In certain embodiments, disclosed herein is a method for making amagnetic silica nanomembrane comprising: a) depositing onto a polymercore having an original size, at least one layer of silicon dioxide; b)depositing onto the polymer core at least one magnetic component; and c)heating the polymer core at a sufficient temperature and time to allowthe polymer core to shrink, and wherein the shrinking of the polymercore creates silica microstructures and/or nanostructures on the surfaceof the silica nanomembrane. In certain embodiments, a method of making amagnetic silica nanomembrane is disclosed herein comprising: a)depositing onto a first side of a polymer core having an original size,at least one layer of silicon dioxide; b) depositing onto a second sideof the polymer core at least one magnetic layer; and c) heating thepolymer core at a sufficient temperature and time to allow the polymercore to shrink, and wherein the shrinking of the polymer core createssilica microstructures and/or nanostructures on the surface of thesilica nanomembrane.

The magnetic silica nanomembranes disclosed herein may be fabricatedusing simple, inexpensive, and/or inventive thermoplastic processes. Insome embodiments, a range of about 2 nm to about 500 nm of silicondioxide is deposited onto about 5 μm to about 500 μm thick polymer core,such as a polyolefin film, by any known means of deposition. Examples ofdeposition methods may include, but are not limited to chemical vapordeposition, electrophoretic deposition, dip-coating, physical vapordeposition, electron beam vapor deposition, sputtering, spin-coating, orliquid phase deposition.

The silica coated polymer film is then heat shrunk in an oven at atemperature sufficient to shrink the polymer. The temperature can varyas a function of the type of polymer used and the starting thickness ofthe polymer. Any heating means can be used, such as, for example,infrared heater, heat gun, or resistive heating element. In certainembodiments, the polymer is heated in a temperature range of betweenabout 100° F. to about 500° F., such as about 250° F. or about 300° F.

The heating time for the shrinking process can also vary as a functionof the type of polymer used and the starting thickness of the polymer.In some embodiments, the polymer may be heated for between about 10seconds to about 10 minutes, such as about 2 to about 3 minutes.

The heat shrinking of the polymer may cause the film to shrink in areaby over 95% in size, while increasing in thickness, and creates ahierarchical structure of microscale folds topped by nanoscale flakes.The magnetic silica nanomembranes can then be fabricated into a varietyof shapes or sizes as needed for various applications. In someembodiments, the polymer core film may shrink to between about 0.1% toabout 95% or about 75% of its original size when subjected to heatshrinking. It is envisioned that the methods disclosed herein may bepracticed on any scale, including small-scale batch processes andindustrial scale roll-to-roll processes.

In certain embodiments, the magnetic silica nanomembranes may be punchedinto circles of varying diameter. In one embodiment, 6 mm diameterpieces can be used, which are capable of fitting into a common 1.5 mLtube. In certain embodiments, 1 mm diameter or smaller pieces can beused, to reduce wetting volume and fluidic dead volume to facilitateextraction of microvolume and low abundance samples. In certainembodiments, the magnetic silica nanomembranes disclosed herein may becapable of binding more than about 150 μg of DNA each, such as more thanabout 160 μg of DNA each, or about 175 μg or more of DNA each. Incertain embodiments, the magnetic silica nanomembranes disclosed hereinmay be capable of binding more than about 250 μg of total nucleic acideach, such as more than about 500 μg of total nucleic acid each, orabout 1000 μg or more of total nucleic acid each. In certain embodimentsdisclosed herein, the magnetic silica nanomembranes may remain stablefor more than at least about six months. In one embodiment, a 6 mmcircle of silica nanomembranes can fit into a cap of a 1.5 mL tube andbe used for nucleic acid separations. These tubes can be premade andavailable as a kit, which may include instructions for use, for example,along with reagents for sample preparation and clean up.

In accordance with certain embodiments, disclosed herein is a method forextracting nucleic acids from a sample comprising: a) obtaining a samplecomprising nucleic acids; b) contacting the sample with a sufficientamount of magnetic silica nanomembrane; c) allowing the nucleic acids inthe sample to adsorb onto the magnetic silica nanomembrane; d) movingthe magnetic silica nanomembrane using a magnet; e) optionally washingthe magnetic silica nanomembrane to remove any non-nucleic acidcomponents; and f) desorbing the nucleic acids from the magnetic silicananomembrane to obtain the isolated and purified nucleic acids from thesample. An exemplary method 300 for the extraction of nucleic acids froma sample are illustrated schematically in the flow chart shown in FIG.3.

In some embodiments, the method for extracting the nucleic acids usingthe magnetic silica nanomembranes disclosed herein may include at stepa) contacting the nucleic acids with a chaotropic agent. This may helpthe nucleic acids to adsorb or bind to the silica microstructures andnanostructures on the nanomembrane. Chaotropic agents or compounds arecompounds that change or disrupt the secondary structure, tertiarystructure, and quaternary structure of proteins, nucleic acids, andprotein-nucleic acid complexes while the primary structure remainsintact. In solution, under chaotropic conditions, the intramolecularinteractions of biological molecules, such as proteins, protein-nucleicacid complexes, and nucleic acids, are disrupted, since chaotropiccompounds interfere with stabilizing intramolecular interactions inbiological molecules, for example hydrogen bonds, van der Waals forces,and hydrophobic effects. Chaotropic compounds usually have large-volumeions that, owing to their size, can interfere with the intermolecularinteractions and reduce the polarity of the solvent as a result, therebydisrupting intermolecular and intramolecular hydrogen bonds.Consequently, many proteins precipitate; however, the helical structureof double-stranded nucleic acid segments is maintained. By addingchaotropic compounds to cell lysates or cell suspensions, proteins canbe precipitated while nucleic acids remain in solution. Under chaotropicconditions, the binding of nucleic acids to silicon dioxide-basedmatrices is greatly favored. Chaotropic compounds comprise, for example,high concentration urea solutions (e.g., 6 to 8 mol/l urea), guanidiniumsalt solutions (e.g., 6 mol/l guanidinium chloride), high concentrationlithium salts (e.g., 4.5 mol/l lithium perchlorate). Chaotropic anionscomprise the anions F⁻, PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, and, for example,BR⁻, I⁻, NO3⁻, ClO4⁻, SCN⁻, and Cl₃CCOO⁻. Chaotropic cations comprisethe cations Li⁺, Mg²⁺, Ca²⁺, Ba²⁺, and for example the guanidiniumisothiocyanate ([CH₆N₃]⁺SCN⁻) and guanidinium chloride. The chaotropiccompounds may also aid in lysing cellular membranes and denaturingproteins.

In some embodiments, the method for extracting nucleic acids using themagnetic silica nanomembranes as disclosed herein further comprises atstep b) contacting the sample with a sufficient amount of magneticsilica nanomembrane in the presence of an aqueous alcoholic solutionsuch as ethanol or isopropanol. It is well-known that the aqueousalcoholic solution may help precipitate the nucleic acids from the othercellular or tissue components in the sample.

In some embodiments, the method for extracting the nucleic acids usingthe magnetic silica nanomembranes disclosed herein may comprise two,three, or more washing steps, such as at step e), for example. Thesewashes can include buffers, alcohols, detergents, or other reagentsknown to be suitable for use in isolation and purification of nucleicacids.

For the purification of DNA, preference may be given to adding RNase ina biologically effective amount to the sample, whereby RNA can bedigested and the intact DNA can be isolated from the sample. The RNasedigestion can be carried out at different times during the extraction,at the earliest after lysis and at the latest after the elution at theend of the purification. However, in certain embodiments, detection ofthe DNA may be in the presence of the copurified RNA, i.e., by omittingthe RNase step or by using buffer conditions that enable selectiveisolation of DNA with exclusion of the RNA.

For the isolation of RNA, preference may be given to adding DNase in abiologically effective amount to the sample. This may result in DNAbeing “digested” and going into solution, while the undigested RNA canbe isolated from the solution. The DNase digestion can be carried out atdifferent times during the extraction, at the earliest after lysis andat the latest after the elution at the end of the purification.

The methods as disclosed herein can be used to enrich a sample in aparticular type of nucleic acid, e.g., DNA or RNA. For example, at stepe), one can add a DNase to remove DNA from the nucleic acids in thesample and enrich the sample in RNA. Likewise, one can add an RNase tothe sample at step e) to remove RNA from the nucleic acids in the sampleand enrich the sample in DNA.

The methods as disclosed herein can be used to enrich a sample in aparticular type of nucleic acid, e.g., DNA, RNA, long nucleic acids, orshort nucleic acids. For example, during the binding step c) and washingstep e), the percentage of alcohol in the buffers can be used to adjustsolubility that will lead to preferred binding and elution of a specificspecies. Salts may also be used to preferentially extract a particulartype of nucleic acid by adjusting the relative solubilities. Organicsolvents may also be used to preferentially extract a particular type ofnucleic acid by partitioning molecules of interest into different liquidphases.

In some embodiments, the method for extracting nucleic acids using themagnetic silica nanomembranes disclosed herein may comprise a dryingstep after step e).

In certain embodiments disclosed herein, the non-porous magnetic silicananomembrane may harbor nucleic acid in a tethered conformation thatenables the extraction of vast amounts of ultra high molecular weight(UHMW) nucleic acid by protecting the nucleic acid against fragmentationand bias binding away from a prone conformation. In contrast tomicroparticles and spin columns, where nucleic acids can be sheared byparticle mixing or by flow through porous matrices, nucleic acidsextracted with the magnetic silica nanomembranes disclosed herein canbind and release directly from the silica lamella without fragmentation.This gentle process ensures rapid isolation (such as, for example, lessthan about 1 hour) of high quality, Mb-sized nucleic acid, with minimalamounts of nucleic acid damage such as nicks and abasic sites, which canbe used to generate high quality, long insert single molecule sequencinglibraries.

For example, in certain embodiments, when the nucleic acids beingextracted are long nucleic acids, the novel tentacle binding methodsdisclosed herein may capture ultra high molecular weight DNA up to amegabase in length and achieve binding capacities up to 1,000,000-timesgreater than silica microparticles. In certain embodiments, the processdisclosed herein may be relatively fast, such as taking less than about1 hour, less than about 45 minutes, less than about 30 minutes, lessthan about 15 minutes, or about 15 minutes. The ultra high molecularweight and low DNA damage that may result from the extraction methodsdisclosed herein may yield high quality sequencing libraries with singlemolecule mean read lengths of at least about 20 kb, such as at leastabout 50 kb, at least about 75 kb, at least about 100 kb, at least about150 kb, at least about 200 kb, at least about 500 kb, or at least about1 Mb.

It will be understood by those of ordinary skill in the art that thenucleic acids that are bound or adsorbed on the magnetic silicananomembranes disclosed herein can be desorbed from the nanomembranes bythe use of any elution solution known in the art. A typical elutionsolution can be a buffer comprising a mixture of (0.5M) ammoniumacetate, 10 mM magnesium acetate and 1 mM EDTA, for example. Anothertypical elution solution can be a buffer comprising a mixture of 10 mMTris base and 1 mM EDTA, for example. Yet another typical elutionsolution can be water.

In accordance with another embodiment, also disclosed herein are methodsfor extracting nucleic acids from formalin fixed paraffin embedded(FFPE) samples comprising: a) obtaining a FFPE sample comprising nucleicacids; b) deparaffinizing the sample; c) contacting the sample with asufficient amount of magnetic silica nanomembranes; d) allowing thenucleic acids in the sample to adsorb onto the magnetic silicananomembranes; e) moving the magnetic nanomembranes using a magnet; f)washing the magnetic silica nanomembranes to remove any non-nucleic acidcomponents; and g) desorbing the nucleic acids from the magnetic silicananomembranes to obtain the isolated and purified nucleic acids from thesample.

In certain embodiments disclosed herein, the FFPE tissue samples may bedeparaffinized by adding an organic solvent, such as xylene. The xyleneis then removed and the sample pellet is washed with graded ethanolsolutions to eliminate xylene and rehydrate the DNA. In otherembodiments, the deparaffinization methods can be varied by altering thexylene concentration, incubation times, and wash protocol to ensure thatall the paraffin is removed and xylene carry through is minimal. FFPEtissue samples may also contain DNA that has been highly cross-linked.In some embodiments, reversal of cross-linking may be performed byheating, such as to a temperature of about 95° C. In other embodiments,this reversal may be performed chemically. After deparaffinization andcross-linking reversal, nucleic acid extraction is performed using aprocess as outlined above.

It will also be understood by those of ordinary skill in the art thatthe compositions, devices, and methods using the magnetic silicananomembranes disclosed herein can be combined with any other analytictechniques useful for isolating, purifying and analyzing nucleic acidsknown in the art.

In accordance with certain embodiments, disclosed herein is a kitcomprising one or more magnetic silica nanomembranes and instructionsfor use of the magnetic silica nanomembranes for isolation orpurification of nucleic acid, such as DNA or RNA, from a sample. Such akit may be provided in a container with other reagents or materialsnecessary to perform the nucleic acid isolation and purification. Thekits disclosed herein can also include a device or apparatus comprisingthe magnetic silica nanomembranes.

In one embodiment, the magnetic nanomembrane is used for DNA extractionfrom a cell sample. The cell sample may first be trypsinized, added to acontainer, and resuspended in a buffer solution, such asphosphate-buffered saline (PBS).

The cell sample may then be lysed. Lysing of the sample may comprisebreaking open of the cells or cellular structures in the sample by anymeans known in the art. Lysis may comprise, for example, mechanicallysis methods such as ultrasound or bead beating, thermal lysis (e.g.,freeze-thaw cycles or heating the sample), and chemical lysis (e.g.,with detergents or enzymes). In certain embodiments, a lysing buffer maythen be added to the cells. In certain embodiments, the lysing buffermay comprise proteinase K, guanidine hydrochloride, and Triton X-100,for example.

After lysis, a magnetic silica nanomembrane as disclosed herein may beadded to the lysed cells to enable nucleic acid binding to the magneticnanomembranes. Additionally, in certain embodiments, isopropyl alcoholmay be added to the solution. In other embodiments, ethyl alcohol may beadded to the solution. After binding, a magnet may be used to pull themagnetic silica nanomembrane to a side of the container such that thelysing buffer and unbound cellular impurities can be pipetted out.

A wash buffer may then added to the container and the magnet released toenable the wash solution to adequately contact the nanomembrane. Incertain exemplary embodiments, the wash buffer may comprise, forexample, isopropyl alcohol or ethyl alcohol. In other embodiments, thewash solution may contain chaotropic salts. The magnet may also be usedto draw the nanomembrane aside to pipette out the wash solution, and thewash may be repeated multiple times, such as two, three, or four times.This series of wash steps may act as stringency washes.

After the final wash, an elution buffer may be added to the containerand the magnet released. In certain embodiments, the container may beincubated to enable the DNA to release, or desorb, from thenanomembrane. Finally, the magnet may again be applied to pull thenanomembrane aside so that the purified DNA may be pipetted out.

The low shear, planar, non-porous format of the magnetic silicananomembranes disclosed herein enables extraction of large genomicfragments with length exceeding about 100 kb, such as greater than about250 kb, greater than about 500 kb, greater than about 1 Mb, greater thanabout 5 Mb, or greater than about 10 Mb.

In certain embodiments disclosed herein, a permanent magnet orelectromagnet may be used.

In some embodiments, the binding and wash buffers may comprise highlevels of isopropyl alcohol (70%) or ethyl alcohol (70%), as well ashigh levels of chaotropic salts such as guanidine hydrochloride (6 M),guanidine thiocyanate (6 M), sodium perchloride (4 M), or sodium iodide(4 M) to facilitate nucleic acid binding to the silica nanomembrane andremoval of contaminating salts, proteins, lipids, etc.

In certain embodiments, the extraction buffers may have components thatare incompatible with the magnetic materials and lead to degradation orleaching into solution. Accordingly, in one embodiment, the extractionbuffers may be optimized to remain chemically compatible with themagnetic materials. In another embodiment, a passivating layer may beused to protect the magnetic material from attack.

In another embodiment, the magnetic silica nanomembrane is used forautomated nucleic acid extraction in conjunction a robotic extractionsystem. Robotic extraction systems may include, for example, pipettingtype instruments such as the Qiagen QIAsymphony, Qiagen Biorobot, TecanFreedom EVO, and Beckman Coulter Biomek workstation and magnetic rodtype instruments such as the Thermo Scientific KingFisher and PerkinElmer Chemagic.

In some embodiments, multiple pieces of magnetic silica nanomembrane maybe used in a single extraction tube.

In accordance with yet another embodiment, the magnetic silicananomembranes can be used in a microfluidic chip format. Themicrofluidic chip is an apparatus that, in certain embodiments,comprises a solid substrate comprising a plurality of discrete magneticsilica nanomembranes regions. The magnetic silica nanomembranes may belocated at spatially defined addresses on the substrate. The magneticsilica nanomembranes may be attached to the chip in a wide variety ofways or confined within certain regions of the chip, as will beappreciated by those in the art. The magnetic silica nanomembranes mayeither be synthesized first, with subsequent attachment or confinementto the chip, or may be directly synthesized on the chip or as part ofthe chip

EXAMPLES Example 1—Magnetic Nanomembrane Fabrication

A 20 μm thick polyolefin film was coated on one side with a 100 nm thicklayer of silicon dioxide using e-beam evaporation. The other side of thepolyolefin film was then alternately coated with a 20 nm thick layer ofsilicon dioxide, a 30 nm thick layer of iron, and a 20 nm thick layer ofsilicon dioxide using e-beam evaporation. The coated polyolefinsubstrate was then heat shrunk in an oven at 300° F. for 3 minutes. Thiscreated a nanomembrane with micro- and nanoscale silica lamella on bothsides. The backside silica lamella comprised a magnetic iron centersandwiched by silica such that magnetic manipulation could be performedand nucleic acids could bind or adsorb to both sides of the magneticnanomembrane. The silica enveloping both sides of the iron acts as abinding surface for nucleic acids and protects the iron from reactingwith buffer components. FIG. 2 is a flow chart illustrating theexemplary fabrication process. FIG. 13 shows an SEM image of the surfacetopology for the magnetic silica nanomembrane, showing the microscaleand nanoscale silica structures, such as flakes, lamella, wrinkles, andfolds. The inset on the right shows a region at a higher magnification.

Example 2—Magnetic Nanomembrane Extraction Process with MCF-7 Cells

The magnetic silica nanomembrane fabricated in Example 1 above was usedfor DNA extraction from 2×10⁶ MCF-7 cultured cells. The cultured cellswere trypsinized and resuspended in a phosphate buffer saline (PBS)solution. A lysing buffer comprising proteinase K, guanidinehydrochloride, and Triton X-100 was then added to the cells andincubated for 1 hour. Then the magnetic silica nanomembrane andisopropyl alcohol were added to the lysed cells to enable nucleic acidbinding to the magnetic silica nanomembrane. A magnet was then used topull the magnetic nanomembrane to the side of the microcentrifuge tubesuch that the lysing buffer and unbound cellular impurities could bepipetted out. FIG. 4 shows a photograph of a magnetic nanomembrane beingpulled to the side of a microcentrifuge tube using a magnetic rack. Awash buffer containing 70% EtOH was then added to the tube, and themagnet was released to enable the wash solution to adequately contactthe nanomembrane. The magnet was then used to draw the nanomembraneaside so that the wash solution could be pipetted out. The wash wasrepeated two more times.

After the third wash, the elution buffer was added to the tube and themagnet was then released. The tube was incubated for 15 minutes toenable the DNA to release, or desorb, from the nanomembrane. Finally,the magnet was applied again to pull the nanomembrane aside, and thepurified DNA was pipetted out. 61 μg of DNA was recovered as measured byPicoGreen® assay. In comparison, DNA extraction of 2×10⁶ MCF-7 culturedcells using phenol-chloroform recovered 45 μg of DNA, a Qiagen MinispinColumn recovered 41 μg of DNA, and DNA extraction from Qiagen magneticparticle technology recovered 49 μg of DNA. FIG. 5 is a bar graphshowing the results of the magnetic silica nanomembrane DNA extractionin comparison to DNA extraction using phenol-chloroform, Qiagen Minispincolumn, and magnetic particle technology. As shown in FIG. 5, themagnetic silica nanomembrane was able to extract a higher yield of DNA.

The purity of the extracted DNA from all of the extraction methods wasmeasured with a spectrophotometer using UV absorbance. The purity of theextracted DNA was comparable for all four methods. The DNA extractedusing phenol-chloroform had high purity with 260/230 and 260/280 ratiosof 1.91 and 2.15, respectively. The DNA extracted using the magneticsilica nanomembrane also had a very high purity with 260/280 and 260/230of 1.96 and 2.16, respectively. The DNA extracted with magnetic beadmicroparticles had a purity with 260/280 and 260/230 of 1.99 and 2.23,respectively, while the DNA extracted with the spin column had a puritywith 260/280 and 260/230 of 1.89 and 2.16, respectively. FIG. 6 is agraph illustrating the UV absorbance versus the wavelength, and Table 1below shows the purity results of the UV absorbance for all four methodsof DNA extraction analyzed.

TABLE 1 Extraction Method 260/280 260/230 Phenol-Chloroform 1.91 2.15Nanomembrane 1.96 2.16 Microparticle 1.99 2.23 Spin column 1.89 2.16

The DNA extracted from the nanomembrane, phenol-chloroform, spin column,and magnetic beads was subjected to pulsed field gel electrophoresis forcomparison of DNA size, and the pulsed field gel electrophoresis imageis shown in FIG. 7. As shown in FIG. 7, the low shear, planar,non-porous format of the nanomembrane enabled extraction of genomicfragments of DNA having lengths exceeding 100 kb with some lengthsexceeding 300 kb.

Example 3—Magnetic Nanomembrane Extraction Process with Whole HumanBlood

A magnetic silica nanomembrane fabricated according to the methoddisclosed above in Example 1 was used to extract DNA from whole humanblood. A lysing buffer comprising proteinase K, guanidine hydrochloride,and Triton X-100 was added to 100 μL of blood and incubated for 1 hour.Then the magnetic silica nanomembrane and isopropyl alcohol were addedto the lysed cells to enable nucleic acid binding to the magnetic silicananomembrane. A magnet was then used to pull the magnetic nanomembraneto the side of the microcentrifuge tube such that the lysing buffer andunbound cellular impurities could be pipetted out. A wash buffercontaining 70% EtOH was then added to the tube, and the magnet wasreleased to enable the wash solution to adequately contact thenanomembrane. The magnet was then used to draw the nanomembrane aside sothat the wash solution could be pipetted out. The wash was repeated twomore times.

After the third wash, the elution buffer was added to the tube and themagnet was then released. The tube was incubated for 15 minutes toenable the DNA to release, or desorb, from the nanomembrane. Finally,the magnet was applied again to pull the nanomembrane aside, and thepurified DNA was pipetted out. The magnetic nanomembrane extracted 18.9μg of total nucleic acid (DNA+RNA). As shown in FIG. 8, a bar graphcomparing the quantity of total nucleic acid extracted from the magneticnanomembrane versus a nonmagnetic nanomembrane, the nucleic acid yieldusing the magnetic nanomembrane is comparable to a non-magneticnanomembrane, indicating that the presence of the magnetic layer doesnot adversely affect the extraction efficiency.

Example 4—Automated Magnetic Nanomembrane Extraction Process withKingFisher™ Duo Prime

Automated extraction was performed on a Thermo Scientific KingFisher™Duo Prime. Rather than using a magnet to hold the magnetic nanomembraneand pipetting solutions in and out of the tube as in FIG. 3, theKingFisher™ Duo Prime extracts DNA using a magnetic head comprising anarray of magnetic rods to transfer magnetic nanomembranes from onesolution to the next as shown in FIG. 11. As illustrated in a method1100 in FIG. 11, actuating the magnetic rod in and out of solutionenables the magnetic nanomembrane to be transferred from one buffer tothe next. Pulling the magnetic rod out of the plastic rod cover enablesthe magnetic nanomembrane to be released into solution to facilitatewashing and mixing. In this manner, DNA extraction can be performed bysuccessively transferring the magnetic nanomembrane into the bindingbuffer to capture the lysed DNA, then into the wash buffers to rinseaway salt and impurities, and finally into the elution buffer to elutethe DNA and remove the used magnetic nanomembrane. The wash steps may beskipped to speed processing. See, for example, FIG. 11.

Automated DNA extraction from MCF-7 cells was performed on a ThermoScientific KingFisher™ Duo Prime. Eight samples were run simultaneouslyusing the robotic extraction system. The cultured cells were trypsinizedand resuspended in a phosphate buffer saline (PBS) solution. As depictedin FIG. 12, the 1×10⁶ cells where placed into seven separate wells ofrow A of a 96 deep well plate 1200. Eight individual magneticnanomembranes were added to wells of row C, wash buffers were added torows D, E, and F, and elution buffer was added to the elution strip.See, for example, FIG. 12. A lysing buffer comprising proteinase K,guanidine hydrochloride, and Triton X-100 was then added to the samplewells (row A) and incubated for 10 minutes with gentle mixing.Isopropanol was manually added to each of the wells in row A. TheKingFisher™ robotic arm comprising a series of magnetic rods thencaptured the magnetic nanomembranes from row C and moved them to row Ato contact them with the sample, enabling the DNA to adsorb the magneticnanomembrane. FIG. 9 is a photograph showing the magnetic nanomembranescaptured by the head comprising an array of magnetic rods on the roboticarm. The robotic arm then moved the magnetic nanomembrane sequentiallythrough wash buffers in rows D, E, and F, as depicted in FIG. 12.

After the third wash, the robotic arm moved the magnetic nanomembrane tothe elution buffer to elute the adsorbed DNA. The magnetic nanomembraneswere then removed by the robotic arm, leaving the purified DNA behind.The amount of DNA recovered from each of the eight samples using theautomated DNA extraction system is shown in the column chart of FIG. 10.The DNA yield using the automated extraction system was 9.1±3.5 μg.

What is claimed is:
 1. A magnetic silica nanomembrane comprising: apolymer substrate having a first surface and a second surface; at leasta first silicon dioxide layer disposed over the first surface of thepolymer substrate; at least a first magnetic component disposed over thesecond surface of the polymer substrate; and at least a second silicondioxide layer disposed over the first magnetic component, wherein themagnetic silica nanomembrane is in a planar conformation, wherein thefirst silicon dioxide layer and/or the second silicon dioxide layercomprises at least one surface morphology chosen from a plurality of (a)microscale silica structures and (b) nanoscale silica structures, andwherein the magnetic silica anomembrane has been heat shrunk.
 2. Themagnetic silica nanomembrane of claim 1, wherein the first magneticcomponent comprises at least one magnetic material chosen fromdiamagnetic materials, paramagnetic materials, ferrimagnetic materials,and ferromagnetic materials.
 3. The magnetic silica nanomembrane ofclaim 1, wherein the first magnetic component comprises at least onemagnetic material chosen from iron, nickel, cobalt, magnetite, hematite,maghemite, steel, alnico, permalloy, alperm, fernico, sendust, andcunife.
 4. The magnetic silica nanomembrane of claim 1, wherein thefirst magnetic component is chosen from at least one layer of magneticmaterial disposed over the polymer substrate, at least one magneticmaterial embedded within the polymer substrate, and at least one polymersubstrate being magnetic.
 5. The magnetic silica nanomembrane of claim1, wherein the first magnetic component comprises a layer of magneticmaterial disposed over the at least one silicon dioxide layer.
 6. Themagnetic silica nanomembrane of claim 5, wherein the at least one layerof magnetic material has a thickness ranging from about 5 nm to about 10um thick.
 7. The magnetic silica nanomembrane of claim 1, wherein thefirst silicon dioxide layer and/or the second silicon dioxide layercomprises at least one microscale and/or at least one nanoscale surfacemorphology chosen from flakes, lamella, wrinkles, and folds.
 8. Themagnetic silica nanomembrane of claim 1, further comprising one or moreadditional magnetic components and/or one or more additional silicondioxide layers disposed over the first silicon dioxide layer and/or thesecond silicon dioxide layer.
 9. The magnetic silica nanomembrane ofclaim 1, further comprising at least a third silicon dioxide layerdisposed between the second surface of the polymer substrate and thefirst magnetic component.
 10. The magnetic silica nanomembrane of claim9, further comprising at least a second magnetic component disposed overthe first silicon dioxide layer disposed over the first surface of thepolymer substrate and at least a fourth silicon dioxide layer disposedover the second magnetic component.
 11. The magnetic silica nanomembraneof claim 1, wherein at least a second magnetic component is disposedover the first silicon dioxide layer disposed over the first surface ofthe polymer substrate.
 12. The magnetic silica nanomembrane of claim 11,wherein at least a third silicon dioxide layer is disposed over thesecond magnetic component disposed over the first silicon dioxide layer.13. The magnetic silica nanomembrane of claim 1, wherein the firstsilicon dioxide layer and/or the second silicon dioxide layer has athickness ranging from about 2 nm to about 500 nm thick.
 14. Themagnetic silica nanomembrane of claim 1, further comprising apassivation layer disposed over the first magnetic component.
 15. Themagnetic silica nanomembrane of claim 1, wherein the first silicondioxide layer and/or the second silicon dioxide layer is deposited usinga deposition method chosen from electron beam evaporation, sputtering,chemical vapor deposition, plasma enhanced chemical vapor deposition,electroplating, atomic layer deposition, chemical solution deposition,and spin coating.
 16. The magnetic silica nanomembrane of claim 1,further comprising a surface functionalization chosen from at least oneof aminopropyl groups, chloropropyl groups, octadecyl groups, octylgroups, quaternary ammonium groups, diethlylaminoethyl group, sulfonicacid groups, phenyl groups, polyethylene glycol, lipids, chitosan,biotin, streptavidin, antibodies, proteins, and enzymes.
 17. Themagnetic silica nanomembrane of claim 1, wherein the polymer substratecomprises at least one thermoplastic material chosen from polymethylmethacrylate, polycarbonate, polystyrene, cyclic polyolefin polymers,polypropylene, polyvinyl chloride, polyethylene, fluorinated ethylenepropylene, polytetrafluoroethylene, and polyvinylidene fluoride.
 18. Themagnetic silica nanomembrane of claim 1, further comprising at least asecond magnetic component disposed between the first silicon dioxidelayer and the first surface of the polymer substrate.
 19. A magneticsilica nanomembrane comprising: a polymer substrate having a firstsurface and a second surface; at least a first silicon dioxide layerdisposed over the first surface of the polymer substrate, the firstsilicon dioxide layer comprising at least one surface morphology chosenfrom a plurality of (a) microscale silica structures and (b) nanoscalesilica structures; at least a first magnetic component disposed over thesecond surface; and at least a second magnetic component disposed overthe first silicon dioxide layer disposed over the first surface of thepolymer substrate, wherein the magnetic silica nanomembrane is in aplanar conformation, and wherein the magnetic silica nanomembrane hasbeen heat shrunk.
 20. The magnetic silica nanomembrane of claim 19,further comprising one or more additional magnetic components and/or oneor more additional silicon dioxide layers disposed over the firstmagnetic component and/or the second magnetic component.